The technique of electroporation (also known as electropermeabilisation, electroinjection or electro-transfection) represents a commonly known means for the inward transfer of membrane-impermeable xenomolecules (e.g. dyes, drugs, hormones, proteins, plasmids etc.) into live cells, or for the controlled release of intracellular substances from the cells. Electroporation has become widely used and recognised because it is more controllable, more reproducible and more efficient than other (chemical or viral) methods of intracellular transport of foreign molecules. The technique of electroporation is based on a temporary increase in membrane permeability which is caused by a reversible electrical break-through of the plasma membrane of cells, with the cells being subjected to high-intensity external electrical pulses of very short duration (field intensities of a few kV/cm, pulse duration of some few 10 μsec). The applied electrical field separates charge carriers via the cell membrane so that a transmembrane potential Vg is induced. It is known that the membrane break-through takes place when the induced membrane-voltage Vg at room temperature reaches a value Vg of approx. 1 volt.
For freely movable suspended cells, the induced membrane voltage Vg linearly depends on the applied field intensity E0 and on the cell radius a, and follows the generally known integrated Laplace equation:Vg=1.5 α·E0·cos θ  (1)wherein θ is the angle relative to the direction of the electrical field. If an average value of e.g. 7 μm is used for the cell radius, the minimum critical field intensity Ekrit which is required for reversible electrical break-through of the membrane regions facing the electrodes (cos θ=1) can be calculated with equation (1): Ecrit=Vc/(1.5·a)˜1 kV/cm. For smaller cells, correspondingly considerably higher field intensities are required to achieve electric break-through through their cell membranes. Thus, for cells with an average radius of e.g. 3 μm, a minimal critical field intensity Ecrit of approx. 2.5 kV/cm is necessary. As soon as the plasma membrane has been permeabilised by the mechanism of electrical break-through, foreign molecules can enter the cell by way of diffusion (or other mechanisms), or intracellular molecules can flow out into the outside medium. The theoretically calculated values for the critical field intensity Ecrit correspond very well with experimental results which were determined in electropermeabilisation tests with the use of fluorescent dyes such as propidium iodide or other smaller reporter molecules.
In practical application, efficient electroinjection of macromolecules (proteins, plasmids, DNA etc.) in freely suspended cells requires significantly higher field intensities than those calculated by means of equation (1). Furthermore, the electropermeabilisation yield can be increased by the use of non-physiological media of low conductivity with a low ion content (and reduced osmolarity). However, both very strong electrical fields and media of low conductivity reduce survivability of the cells. In the case of rare or valuable cells (for example genetically modified hybridoma cells or dendritic cells) this can in turn significantly reduce the number of available cells. For this reason, very careful and time-consuming optimisations of the electropermeabilisation protocols are necessary for suspension cells.
The effect of electroporation on cell membranes can be determined directly by measuring their electrical resistance (or the impedance) by means of intracellular electrodes. However, in the case of electroporation of suspended cells, this method is limited to cells of sufficient size (e.g. giant algae, xenopus oozytes) and cannot be used on most cells of animal or plant origin which are only a few micrometers in size. The application of extracellular electrodes for measuring the impedance of cell suspensions requires very high cell density (i.e. 30-90% cytocrit value (=cell content of the total suspension)). This method cannot be used for carrying out measurements in diluted cell suspensions (cytocrit<1%), as provided in the electroporation of suspended cells.
It is known to carry out electroporation on embedded cells which are embedded in, or arranged on, micropores of a fixed carrier element made of an electrically insulating material. Electroporation of solid-phase adsorbed cells has the advantage that the electrical flux lines between two electrodes, arranged at opposite sides of the carrier element, are forced to flow through the pores of the carrier element and thus through the cells. From practical application, devices for electroporation of solid-phase adsorbed cells are known, manufactured by Equibio of Great Britain.
For example a device for electroporation and electrofusion of adsorbed biological cells is described in WO 93/02178. A cylindrical chamber is provided which is divided into two compartments by an electrically insulating membrane with through-pores. If pressure is exerted on the liquid, suspended cells are held in place in or on the pores, and exposed to electrical fields which are generated with electrodes in the compartments. While this technique makes possible electroporation and electrofusion in solutions of high or low conductivity, it has, however, the disadvantage in that the arrangement of the cells on the carrier element and the result of electrical treatment cannot be observed or monitored. The device known from WO 93/02178 is thus unsuitable for the treatment, in particular, of rare or valuable cells under practical conditions. The known device has a further disadvantage in that the chamber is combined with a closed pressure system. The design of the device is expensive and handling in practical application is complicated.
Electroporation of individual biological cells in Microsystems and in particular the introduction of an impedance technique are described by Y. Huang et al. in “Sensor and Actuators A”, volume 89, 2001, pages 242 ff, and in U.S. Pat. No. 6,300,108 B1. In an electroporation chip which has been produced on the basis of semiconductor materials, the cell is held, by means of liquid pressure, on a connection aperture in a membrane-shaped wall between two compartments. In each of the compartments, electrodes are provided by means of which a poration field can be generated which permeates the fixed cell. Y. Huang et al. describe that the current flow through the carrier element changes, depending on whether the connection aperture is open or occupied by an intact or a permeabilised cell. In order to detect electroporation, the direct current is measured and current-voltage ratios are determined during generation of the poration field, i.e. during permeabilisation of the cell. The poration result can be optically monitored by means of a transparent top of the poration chip.
While the system described by Y. Huang et al. allows real-time monitoring of the poration process, it is, however, not suitable for practical applications in which a multitude of biological cells are to be treated. Furthermore, direct current measuring only provides an information about the membrane characteristics during electroporation, without allowing any further-reaching characterisation of the treated cell for any subsequent processing steps.
Improvements to the poration chip of Y. Huang et al. are described in WO 01/07585, WO 01/07584 and WO 01/07583. These improvements relate in particular to the possibility of treating a multitude of biological cells at the same time. The technique described in the above-mentioned WO publications has the following disadvantages. For real-time monitoring of the poration results, a current measurement is provided at the same time as electroporation. Two measurement and two poration electrodes are provided in the poration chip, thus complicating the design of the poration chip. Real-time monitoring of the poration result covers only commencement of poration and recognition of irreversible damage to the cells. However, this information is insufficient for practical electroporation applications, for example in the field of medicine. Further characterisation of the cells, either before or after electroporation, has to take place with optical means. Optical observation, however, only produces qualitative assessments.
Generally, there is a problem in that only the current is measured in conventional techniques. Electrical measurements supply information that can be evaluated only to a limited extent. There is no specification of measuring parameters that furnish reliable measuring results. However, the measuring object comprising electrolyte and biological cell in the measuring chamber is a complicated structure characterised by capacities and resistances which, depending on the type of cells to be treated, requires other measuring parameters.